This invention relates to methods designed for use in the analysis, assay, or location of proteins, polypeptides) and other substances of biological interest by linking (xe2x80x9clabellingxe2x80x9d) them to another molecule or molecules which can take part in a reaction resulting in the emission of light. The labelled substance, to be termed xe2x80x9cluminescent reagentxe2x80x9d, may be used in various types of biological investigations, preferably immunoassays and protein binding assays. The amount of the xe2x80x9cluminescent reagentxe2x80x9d is measured by recording the light emitted after producing the appropriate conditions required for the luminescent reaction to take place. Here, the luminescent reaction is one which generates chemiluminescence which is distinguished from fluorescence or phosphorescence. The use of the xe2x80x9cluminescent reagentxe2x80x9d enables the analysis, assay, or location of proteins or other substances of biological interest to be carried out without experiencing any of the disadvantages associated with the use of radioactive labelled substances such as are commonly used at present. These disadvantages are as follows. Firstly, the formation of such a radioactive reagent involves the use of highly radioactive and hence potentially hazardous species. Secondly, the shelf-life of such a radioactive reagent is often short because of its continuous decay. Further, the emission of radiation from the radio-isotope may be deliterious to the substance to which the radio-isotope is linked. Thirdly, it is often difficult to label proteins sufficiently to provide a sensitively and rapidly detectable reagent.
In contrast, the xe2x80x9cluminescent reagentxe2x80x9d is non-radiotoxic, stable, and can be Quantified both rapidly and in small amounts.
In a preferred form the invention comprises a compound consisting of aryl acridinium ester, linkage and N-succinimidyl ester moieties. This compound undergoes a light emitting reaction in the presence of a dilute aqueous solution of sodium hydroxide and hydrogen peroxide and the amount of compound can be quantified by measurement of the intensity of the emitted light or rate of photon emission. Further the compound reacts with other substances containing primary and second aliphatic amines to yield chemiluminescent derivatives. The compound has the advantage of being stable under normal storage conditions and of reacting with the aforementioned amines under mild reaction conditions.
The compound can be represented by the general formula: 
in which X is an anion, R1 represents H, C1-C10 optionally substituted alkyl, alkenyl, alkynyl or aryl, R2, R3 are preferably, hydrogen, amino, substituted amino, carboxyl, hydroxyl, alkoxyl, nitro-, or halide substituents, and R4 is preferably an optionally substituted phenoxy-moiety. According to a preferred feature of the invention an ester linkage exists between the acridinium and phenyl moieties as exemplified in the following formula: 
where R5 comprises one of the following: 
xe2x80x94halidexe2x80x83xe2x80x83(e)
xe2x80x94azidexe2x80x83xe2x80x83(f)
where R6 represents groups such as R1 and Y represents a halide. Preferably R5 is linked to the phenyl residue via carbon, nitrogen or oxygen containing groups which are optionally substituted by substituents of a hydrophilic nature.
From another aspect of the invention the labelling compound is prepared by reacting an acridine with a cyanide, and thence converting the product via a carboxy derivative to a halocarbonyl acridine derivative. The acridine derivative may be further reacted with a phenolic moiety substituted with one of the groups defined above as R5, and the product may be methylated at position 10. In some cases the R5 moiety, or part thereof, or the linking Croup is protected before methylation, for example by amidation, esterification or acylation.
Thus in a particular example a N-hydroxysuccinimidyl ester is synthesised via the following route. Acridine is reacted with potassium cyanide and benzoyl chloride so as to yield a 9-cyano-derivative which is subsequently purified by recrystallisation. The material so obtained is reacted with sulphuric acid, thence sodium nitrite to yield the 9-carboxy-derivative which is purified, dried and reacted with thionyl chloride to give the 9-chloro-carbonyl-derivative. This is allowed to react with N-succinimidyl 3-(4-hydroxyphenyl) propionate to yield the corresponding ester of 9-carboxyacridine. The product is methylated at the 10-position, preferably using dimethyl sulphate or methyl pluorosulphonate to yield the final chemiluminescent product which is isolated by trituration with ether followed by filtration and dessication. An alternative method involves the formation of 9-chlorocarbonyl acridine as described above thence formation of the- corresponding methyl ester using methanol followed by methylation at the 10-position preferably using dimethyl sulphate or methyl fluorosulphonate. This is then converted to the corresponding carboxylic acid by hydrolysis thence to the corresponding acyl chloride by reaction with thionyl chloride. The product of this reaction is then mixed with N-succinimidyl-3(4-hydroxyphenyl) propionate to yield the final product which is isolated by trituration.
According to a further aspect of the invention a luminescent labelling material comprises an acridinium compound as defined, linked to a protein or polypeptide or other organic substance of biological interest to provide a stable immunologically reactive luminescent reagent. The compounds represented by formulae(1) and (2) above are capable of reacting with proteins at temperatures between 0 and 40xc2x0 C. under aqueous conditions optionally in the presence of organic solvents to yield xe2x80x9cluminescent reagentsxe2x80x9d which are preferably stable and immunologically active. The reaction which results in the formation of the xe2x80x9cluminescent reagentxe2x80x9d is complete preferaby within 30 minutes, more preferably within 5 minuts, such that at least one mole, and preferably at least three or ten moles of chemiluminescent compound are incorporated into each 1 mole of protein, excess chemiluminescent label is inactivated with lysine which also acts to dissociate non-covalent interactions between chemiluminescent compound and protein.
Preferably the labelled reagents are antibodies more preferably monoclonal antibodies which, when labelled, can be purified so as to be free of excess chmiluminescent compound by gel permeation chromatography preferably in a buffer system containing non-immune gamma globulins.
From a further aspect the invention consists in an assay procedure in which a luminescent labelling compound as defined above is linked with a substance of biological interest, a light emitting reaction is triggered and the amount of the substance is quantified or detected by measuring or sensing the emitted light. The invention is especially applicable to immuno assy, and preferably two-site immunometric assays for the quantitation-of polypeptide antigens. Here a solid phase derivative of a polyclonal or monoclonal antibody, which may be an antibody coated tube, microtiter plate well or particulate solid phase, preferably a cellulose derivative, is used to bind antigen molecules. The uptake of antigen, which depends on the antigen concentration, is measured by allowing the resultant solid phase to come into contact with the labelled reagent and measuring the luminescence activity associated with the solid phase following its separation from the assay mixture. Such an assay is preferably performed by adding the solid phase antibody and the xe2x80x9clabelled reagentxe2x80x9d simultaneously to the antigen containing solution thence allowing the immunological reaction to proceed, preferably for 4 hours, more preferably for 1 hour, before isolation and luminometric measurement of the solid phase. Alternatively the antigen amy be reacted first with labelled antibody for a short period of say xc2xd to 1xc2xd hours and then subsequently with solid phase antibody for a similar period. After isolation of the solid phase, the luminescence activity associated with the solid phase is quantified.
Alternatively indirect determination of the antigen concentration may be performed by using a labelled second antibody which selectively binds the soluble antibody used initially, that is, using unlabelled soluble first antibody. Such a labelled anti (globulin) antibody has the advantage of being a universal labelled reagent.